Helicobacter Pylori Different Virulence Genes and the Risk of Gastric Cancer in Iranian Patients

Background: Some disease-specic Helicobacter pylori virulence genes can be used for predicting the outcome of diseases. Thus, the current study aimed to explore the frequency of the vacA, cagA, sabA, dupA, babA, oipA, and iceA1 genes in H. pylori isolates, and to determine whether any association exists between the expression of these genes and gastric cancer (GC). Methods: H. pylori isolates were collected from individuals with digestive diseases. The cagA, vacA, dupA, sabA, babA, oipA, and iceA1 genes were determined by PCR. The qRT-PCR was used for expression analysis of the tested genes. Results: The presence of the cagA, vacA, dupA, sabA, babA, oipA and iceA1 genes in H. pylori isolates were 41.3%, 95.5%, 31.0%, 93.1%, 55.1%, 82.7%, and 62.0%, respectively. The cagA, dupA, and babA expression in the GC patients was statistically higher than that of the control group (P <0.05). Conclusions: Our results indicated a high diversity and frequency of H. pylori virulence genes among individuals with gastric diseases in the Iranian population. The cagA, dupA, and babA expression were signi�cantly higher in the GC patients, thus, it may suggest that screening of these genes may help identify peoples at higher risk for GC.


Background
Gastric cancer (GC) is a global concern and remains the third leading cause of mortality from cancer worldwide [1].Positive serologic tests for Helicobacter pylori is among the main causes of GC, accounting for approximately 89% of GC cases worldwide [1][2][3].The relationship of H. pylori infection and several extra-intestinal diseases, such as migraine, Alzheimer's, and mild cognitive impairment, has also been addressed [4][5][6][7][8].
Earlier surveys indicated that those with chronic infection with H. pylori were more likely to develop GC [9][10][11].Moreover, the World Health Organization and the International Agency for Research on Cancer (IARC) categorized H. pylori as a Group 1 carcinogen for humans [12].
These virulence genes may lower the threshold for neoplastic alteration by dysregulating the host intracellular signaling pathways [13].
In Iran, the rate of H. pylori infection is very high lies between 60% and 90% in different ages and geographic regions [21].Likewise, the majority of H. pylori-infected subjects are cagA-positive strains that are related to a greater risk of GC in some Iranian populations [21].Despite the high prevalence of cagA positive H. pylori in Iran, its contribution to excess cancer risk is unclear.Furthermore, limited studies in Iran have concurrently investigated the relationship to GC of these virulence-related genes.Thus, in the current study, we examined the association of cagA, vacA, dupA, sabA, babA, oipA, and iceA1 genes of H. pylori with GC.

Methods
Study population, samples, and culture Gastric biopsies for isolation of H. pylori were taken from subjects with digestive diseases at a teaching hospital in Qom, Iran in the year 2018.Eleven biopsy specimens including four specimens of the greater and lesser curvature of the antrum and four specimens of small and large curvature of the gastric body and two specimens of the duodenum (5 biopsies for histopathological tests and 5 biopsies for culture and molecular tests) and one specimen for testing Rapid urease RUT was taken from each patient.
The culture method was performed as described by Dabiri et al, previously [22].Brie y, each specimen was placed into brucella Agar with 10% de brinated sheep blood containing the supplement (5 mg/l amphotericin B, 5 mg/l trimethoprim, 10 mg/l vancomycin).The plates were placed at 37 °C (CO2 10%, N2 85%, O2 5%) and were studied after 5 days of incubation.Urease, catalase, oxidase tests were used for con rmation of H. pylori isolates.The colonies of H. pylori were further used for molecular analysis.

Commercial Rapid Urease Test
The RUT was done as described by the instructions of the manufacturer (HelicotecUT® Plus, Taiwan).The tests were recorded and observed within 1 hour.The color changed from yellow to pink indicated a positive RUT.

Molecular Identi cation Of H. Pylori
DNA was extracted using a commercial kit (Bio Basic, Canada).For molecular con rmation of H. pylori isolates, PCR for 16SrRNA and glmM genes using speci c primers was performed based on published papers [23].

Results
In this study, 190 patients with digestive disorders were enrolled.Among them, 13 were excluded from the study due to the unavailability of demographic information.Based on histological and endoscopic ndings, patients were classi ed into three groups: 91 people without ulcer (NUD = non-ulcer dyspepsia), 55 with peptic ulcer disease (PUD), and 31 with GC.According to the RUT, 51 (28.8%) tissue samples were positive for H. pylori and 126 (71.2%) specimens were negative.In the histology method, 40 (22.6%)biopsy specimens were positive and 137 (77.4%) specimens were negative.Likewise, in the culture method, H. pylori-positive and -negative samples were 42 (23.7%)and 135 (76.3%) respectively.Among the specimens with a positive culture, 13 samples were excluded from the study due to a low number of bacteria and insu cient amounts of DNA and RNA for molecular tests.Finally, 29 isolates of H. pylori remained for further analysis (Fig. 1).
The patients with positive culture were assessed for the relation of gender, age, ethnic group, history of smoking, and alcohol intake with the severity of illness (Table 2).No statistically signi cant difference was observed between these variables and gastroduodenal diseases.However, the age groups of > 60 years had an increased rate of GC disease.

Pcr Results Of The Tested Genes
The presence of the cagA, vacA, dupA, sabA, babA, oipA, and iceA1 genes was investigated in patients with a positive culture for the H. pylori.There was no statistical relationship between these genes and clinical Consequences (Table 3).The cagA and vacA genes were detected in 41.3% (12/29) and 96.5% (28/29) of the patients, respectively.Similarly, to vacA.sabA, and oipA were found more commonly in patients with H. pylori.Of the 29 H. pylori-infected samples, dupA, babA and iceA1 were detected in 31.0%(9/29), 55.1% (16/29), and 62.0% (18/29), respectively.As shown in Table 3, cagA, dupA, and babA were most commonly found in the GC patients, whereas sabA, oipA, and iceA1 were most commonly found in the Non-ulcer patients (p > 0.05).Although, the vacA gene was frequently found in all patient groups; there was no signi cant difference among the investigated patient (p > 0.05) (Table 3).

H. Pylori Virulence Gene Expression
Expression of the Virulence-associated genes cagA, vacA, dupA, sabA, babA, oipA, and iceA1 was detected using qRT-PCR.Expression was calculated based on the cycle threshold for the gene of the target relative to that for the 16srRNA (control group).As shown in Fig. 2A, cagA expression in the GC group was 2336-fold higher than that of the control group, which was statistical signi cance (P < 0.0001).
Likewise, dupA and babA expressions in the GC group were statistically 378-and 3.4-fold higher than that of the control group, respectively (p < 0.05) (Fig. 2B, C).On the contrary to the cagA, dupA, and babA, the expression of vacA and sabA genes in the GC group were signi cantly 1.6-and 4.5 times lower than that of the control group, respectively (p < 0.05).For oipA and iceA1 genes, there was no signi cant difference between the GC and control groups (p > 0.05).

Discussion
In the current study, we indicated the association between virulence genes of H. pylori and the GC.H. pylori infections are frequently asymptomatic and only a few individuals develop GC [31].It has been indicated that some H. pylori virulence genes may play a signi cant role in the progress of post-H.pylori infection disorders such as GC.
In the previous studies, cagA and vacA of H. pylori were shown to have a signi cant association with peptic ulcers and GC [32][33][34][35][36]. Nevertheless, other studies have not established this association, specifying geographic variation [37].Based on our analysis, the cagA gene was observed in 41.3% of the H. pylori-infected patients.The current reports are in comparison with earlier reports that show the cagA gene was present in about 65% of H. pylori isolates obtained from Asian and Western peoples [37][38][39].
The cagA-positive isolates of H. pylori have been also reported to be associated with gastric mucosal atrophy and GC [35,40].In a meta-analysis study, it was shown that patients infected with cagA-positive H. pylori isolates have an increased risk for GC [32].Similarly, the subsequent analysis showed that infection by cagA-positive H. pylori isolates can increase the risk of GC [35].Gohardani et al. showed that the expression of cagA was higher in Iranian patients with peptic ulcers compared to those with non-ulcer dyspepsia [41].However, in studies conducted in Korea and Japan, there was no signi cant association between the cagA gene and the severity of gastroduodenal diseases [42,43].According to recent studies, the diversity of cagA types may explain such discrepancy [37].
The expression of babA and dupA genes were also signi cantly associated with GC in the current study.
Gerhard et al., and Hocker et al. revealed that H. pylori strain to carry babA was associated with the highest risk of the ulcer [44,45].In our results, however, there was not any association between babAand dupA-positive H. pylori isolates and the development of GC, even though the expression of babA and dupA genes were also signi cantly associated with GC.
Our ndings were inconsistent with the dupA gene being a marker for intestinal metaplasia and GC.In this survey, there was not any association between the presence of dupA and GC.Hong Lu et al. indicated that dupA was associated with a reduced risk for GC and an increased risk for duodenal ulcers [46].A study in Brazilian adults also described that the existence of dupA was statistically lower in patients with GC [47].However, Argent et al., reported that the dupA-positive H. pylori strains were more frequent in GC patients than duodenal ulcers (71% vs. 50%) [48].The difference between these studies may be related to differences in the descriptions of geographic variations of circulation strains or patient groups.
In the current study, the vacA gene was present in 96.5% of H. pylori isolates, but no signi cant association between this gene and GC was observed.Likewise, in Asian studies, the existence of vacA was not statistically associated with GC [42,43].
The iceA1 gene was not associated with GC in this study.However, some reports have proposed a reverse correlation [37,38].Two distinct allelic variants of iceA1 exist, in which iceA1 has been suggested to be associated with gastric disease [37].
The oipA gene was existing in 82% of H. pylori isolated in the current which was following the previous studies [49,50].Kudo et al. and Yamaoka et al. also identi ed the oipA gene from 30% and 45.9% of H. pylori isolates, respectively [51,52].Similar to our result, Shao et al., declared that no relationship exists between the oipA gene and gastrointestinal diseases [53].Overall, the association between the oipA gene and GC needs further investigation.
In our study, the expression of some virulence genes of H. pylori was signi cantly higher in GC patients.Thus, it has been revealed that the eradication of H. pylori in patients with early GC would prevent the progress of new cancer [54].

Conclusion
Our nding indicated a high diversity and frequency of H. pylori virulence genes in GC patients in the Iranian population.Although there was not a signi cant association between different virulence genes with the clinical outcomes, the cagA, dupA, and babA expression in the GC group was signi cantly higher than that of the control group.Thus, it may suggest that screening of these genes may help identify populations at higher risk for GC.
Samples included in the study.
Samples included in the study.

Table 1
Primer sequences of the tested genes Rerfvers.
for 30 s, and 72 °C for 25 s.The reactions were carried out using an ABI step ONE Plus thermal cycler.A 2% agarose gel was used for the analysis of products.Statistical AnalysisData analysis was carried out using Statistical Package for the Social Sciences software (SPSS version 23.0) and Graph Pad Prism software version 8.0.2.A Chi-square test was used for the analysis of categorical data.ANOVA statistical test and Tukey post hoc test were applied for quantitative analysis.P values of less than 0.05 were considered statistically signi cant.

Table 2
Distribution of 29 patients with different clinical outcomes, according to age, gender, ethnic group, history of smoking, and alcohol intake.

Table 3
The cagA, vacA, DupA, SabA, BabA, OipA, and iceA1 status of H. pylori isolates obtained from 29 patients with different clinical outcomes.